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Biomedical subjects

C W Lin

Publications and source records attributed to C W Lin.

At least 73 records · Page 4Linked to original sources
PubMed1995

Sampling variation caused by A/D cards due to external trigger.

In electrophysiological recording, a microcomputer-based analog-to-digital (A/D) card is an indispensable instrument for signal acquisition and analysis. In our studies, evoked responses sampled by our A/D card showed variation among different cycles. If several cycles had been averaged, the resultant waveform would have a smaller peak amplitude and a longer duration. To explain this phenomenon, a simulated sampling model of compound action potential was proposed. Our experimental data agreed very well with the prediction of the simulated model. The long and varied delay time between the external trigger and the first sampling in each cycle by our A/D card might be the main cause of such variation. This problem could not be solved by any post-sampled programming. Hence, for those electrophysiological laboratories which sampled evoked responses, to buy a new A/D card might be the most straightforward solution to the problem.

Action Potentials↗
PubMed1995

Function of human Fc gamma RIIA and Fc gamma RIIIB.

Fc gamma RIIA (CD32), a conventional type I transmembrane protein, and Fc gamma RIIIB (CD16B), which has a glycan phosphatidylinositol (GPI) membrane anchor, are both expressed on human neutrophils. Although some details remain to be elucidated, signaling following crosslinking of Fc gamma RIIA requires the activation of tyrosine kinases of both Src-family kinases and Syk, resulting in tyrosine phosphorylation of Shc, phospholipase C gamma isozymes, and a [Ca2+]i transient. Ligation of neutrophil Fc gamma RIIIB triggers a [Ca2+]i transient, and degranulation, although probably not ADCC or an oxidative burst. However, the mechanism for signal transduction by Fc gamma RIIIB, which lacks a transmembrane domain, is not known. Fc gamma RIIA and Fc gamma RIIIB appear to synergize with each other, leading to suggestions that the GPI-anchored Fc gamma RIIIB utilizes the Fc gamma RIIA signaling apparatus. The relevance of proposed specialized membrane domains enriched in GPI-anchored proteins, sphingomyelin and glycolipids to the signaling properties of Fc gamma RIIIB likewise remains to be explored.

Gene Expression Regulation↗
PubMed1995

Intestinal ammonium transport by ammonium and hydrogen exchange.

BACKGROUND: Ionized ammonia (NH3) transport in the intestine has not been previously established as a mechanism of acidosis in urinary intestinal diversion or hepatic failure. STUDY DESIGN: The purpose of this study was to establish that ionized transport of ammonium (NH4) occurs in the intestine and to characterize the mechanism of its transport using the methodology of brush border membrane vesicles and acridine orange fluorescence. RESULTS: An NH4/H exchange was demonstrated and found to be the dominant mechanism causing a pH change when NH4 is transported across the lumenal membrane. Ionized NH4 transport was demonstrated to occur against an NH3 concentration gradient. The Km was 1.02 mmol and the Vmax was 247 U/sec. The Hill coefficient was 0.97, indicating a single port. Ammonium hydrogen exchange could be inhibited by amiloride but not by bumetanide. Sodium, potassium or chloride, or both, did not effect the NH4/H exchanger. CONCLUSIONS: This study establishes that ionized NH4 transport occurs across the small intestine brush border in exchange for a hydrogen ion.

Acridine Orange↗
PubMed1995

Characterization of cloned human dopamine D1 receptor-mediated calcium release in 293 cells.

Dopamine (DA) D1 receptors are generally known to couple only to Gs and cAMP production. Recently, D1 receptors expressed in mouse Ltk- cells have been shown to induce cAMP production, phosphoinositide (PI) hydrolysis, and calcium mobilization [Mol. Endocrinol. 6: 1815-1824 (1992)]. To further evaluate second messenger systems that could be activated by the D1 receptor, we examined the effects of DA, (R)-(+)-SKF-38393, and DA antagonists on cAMP production and calcium release in human embryonic kidney 293 cells stably expressing three different levels (Bmax = 0.12, 1.4, and 23 pmol/mg of protein) of the human D1 receptor. DA and (R)-(+)-SKF-38393 activated cAMP production and calcium release in all three D1-293 clones, and their potency was proportional to receptor density. The efficacy of SKF-38393 was also increased with receptor density in both cAMP and calcium studies. The effect of DA on calcium release consisted of a transient peak response (< 20 sec) that declined to an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-sensitive plateau level above the base-line (>5 min). The effect of DA on cAMP and calcium release was selectively inhibited by SCH23390, a selective D1 antagonist, and not by spiperone, a selective D2 antagonist. DA did not induce PI hydrolysis in any of the three receptor-expressing clones. A 24-hr pretreatment with cholera toxin (2 micrograms/ml) greatly attenuated the effect of DA on cAMP formation and calcium release. To address how DA could activate calcium release without enhancing PI hydrolysis, the effects of forskolin, thapsigargin, and isoproterenol (Iso) were studied. Similarly to the effects of DA, forskolin and Iso stimulated cAMP production and calcium release from D1-293 cells. Cells that were stimulated with Iso or forskolin showed a reduced response to subsequent addition of DA. Pretreatment of D1-293 cells with thapsigargin, a selective Ca2+-ATPase inhibitor, elicited calcium release from the inositol-1, 4, 5-trisphosphate-sensitive calcium store and attenuated the response to subsequent addition of DA. Carbachol stimulated PI hydrolysis and calcium release but had little effect on cAMP production. Prestimulation with carbachol abolished the calcium response to DA, Iso, or forskolin. These studies indicate that D1 receptor-mediated calcium mobilization in 293 cells is dependent on cAMP production and the cAMP-dependent calcium store is part of the inositol-1,4,5-trisphosphate-sensitive calcium pool.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1994

Influence of dye and protein location on photosensitization of the plasma membrane.

Two membrane-photosensitizing dyes were used to investigate whether selected sites in the plasma membrane vary in their sensitivity to damage by singlet oxygen (1O2*) and, if so, what factors are responsible for the variation. The relative ability of Rose bengal (RB) and merocyanine 540 (MC540), both of which localize in the plasma membrane and produce 1O2*, to photosensitize five plasma membrane functions in P388D1 cells was evaluated. The five membrane functions assessed were: plasma membrane potential, proline transport, facilitated glucose diffusion, 5'-nucleotidase activity, and dye exclusion. Photosensitization efficiency by RB varied by a factor of 188 for these membrane functions, whereas for MC540 a range of only 24 was found. RB was a more efficient photosensitizer than MC540 but the relative efficiencies varied with the membrane function. The wide range of P50 values for RB suggests that it binds selectively to membrane sites where it causes damage with high efficiency; possibly a non-1O2* mechanism is involved. In contrast, MC540 photosensitized the three membrane functions involving integral membrane proteins about equally suggesting that differences are due to small variations in the distribution of MC540 in the plasma membrane and/or variations in the inherent reactivity of the membrane targets with 1O2*. The results indicate that the lability of membrane sites to photosensitization depends both on their inherent reactivity with 1O2* and the relative location of specific protein and dye molecules.

5'-Nucleotidase↗
PubMed1994

Novel Asp32-replacement tetrapeptide analogues as potent and selective CCK-A agonists.

A series of novel CCK tetrapeptide analogues of the general formula Boc-Trp-Lys(Tac)-N(R)-(CH2)nCON(R')Phe-NH2 (Tac = o-tolylaminocarbonyl), where R,R' = H or Me and n = 1-5, have been synthesized and tested. These analogues, which lack an acidic residue at the penultimate position, demonstrated surprisingly high CCK-A receptor affinity and selectivity. The effect of N-methylation pattern on CCK-A receptor affinity showed consistent trends for analogues in which n = 1, 2, or 3, with the di-N-methylated analogues having the highest affinity in each case. However, none of these analogues had full agonist activity, as measured by percent maximal PI hydrolysis. Two conformationally constrained analogues also demonstrated high CCK-A receptor affinity and selectivity, as well as nearly maximal agonist activity. In addition, one of these conformationally-constrained analogues demonstrated anorectic activity in rats.

Amino Acid Sequence↗
PubMed1994

Changes in energy metabolites, cGMP and intracellular pH during cortical spreading depression.

The spatial and temporal distribution of cerebral metabolites and pHi were examined in the cortex during spreading depression. Acidification and marked depression in the energy status of the tissue was evident at the wavefront of spreading depression. In its aftermath, the residual activation of glycolysis and accumulation of cGMP persisted for minutes after a relatively rapid restoration of high-energy phosphates and pHi.

Adenosine Triphosphate↗
PubMed1994

Evidence that the guanosine substrate of the Tetrahymena ribozyme is bound in the anti conformation and that N7 contributes to binding.

The group I self-splicing introns are RNA enzymes that catalyze phosphodiester-exchange reactions. These ribozymes have a highly specific binding site for guanosine, a substrate for the first self-splicing reaction (Bass & Cech, 1984). The binding site for guanosine has been localized to a specific region of the ribozyme (Michel et al., 1989), but the conformation of the bound guanosine substrate remains unknown. Most analogs of guanosine with substituents at C8 have a preference for the syn conformation; however, some C8-substituted analogs have the potential to form a hydrogen bond between the C8 substituent and the 5'-hydroxyl that would stabilize the anti conformation; we have found that analogs with the potential to form such a hydrogen bond are more active substrates than those that cannot form such a hydrogen bond. These observations led us to test 8-5'-O-cycloguanosine, which is locked in the anti conformation, and 8-(alpha-hydroxyisopropyl)guanosine, which is locked in the syn conformation; the former is active as a substrate, while the latter is inactive. These results strongly suggest that guanosine is bound to the ribozyme in the anti conformation and provide an additional constraint on structural models of this RNA enzyme. We have also examined a series of N7-substituted guanosine analogs; this position had previously been assumed to be unimportant for substrate binding since 7-methylguanosine is an excellent substrate. However, we have found that 7-deazaguanosine and 7-methyl-7-deazaguanosine are less active substrates than guanosine. We discuss several models for the role of N7 in guanosine binding.

Animals↗
PubMed1994

Tetrapeptide CCK-A agonists: effect of backbone N-methylations on in vitro and in vivo CCK activity.

N-Methylation of backbone amide bonds was conducted on a tetrapeptide that had been identified previously (Shiosaki, K.; et al. J. Med. Chem. 1991, 34, 2387-2842) as a potent and selective CCK-A agonist. N alpha-Methylation at the position corresponding to Asp32 (CCK-33 numbering) was consistent with high affinity, efficacy, and selectivity for the CCK-A receptor. Combination of this (N-Me)Asp with the (N-Me)Phe modification also provided a highly active analogue. The observation of parallel structure-binding affinity profiles with respect to sites of N-methylation in the C-terminal regions of tetrapeptide vs heptapeptide CCK analogues suggests that the two series interact similarly with the CCK-A receptor.

Amino Acid Sequence↗
PubMed1994

Tetrapeptide CCK agonists: structure-activity studies on modifications at the N-terminus.

We had reported earlier on a novel series of potent and selective tetrapeptide cholecystokinin-A (CCK-A) agonists of the general structure Boc-Trp-Lys[epsilon-Y]-Asp-N(R)PheNH2 [Y = amides, ureas; R = H, Me] that were potent anorectic agents in rats. In an effort to optimize the potency, selectivity, stability, and efficacy of our lead candidate A-71623 [R = Me, Y = o-tolylaminocarbonyl; Tac] toward development of a clinical candidate, we have explored a series of analogues in which the N-terminal Boc functionality was systematically replaced with various amides, ureas, carbamates, and sulfonamides of differing size, hydrophobicity, and stereoelectronic properties. In general, these analogues maintained good potency and selectivity for the CCK-A receptor (guinea pig pancreas), as well as potent anorectic activity in rats. Those analogues exhibiting equal or superior activity compared to A-71623 but differing physicochemical properties may represent superior drug candidates.

Amino Acid Sequence↗
PubMed1994

Enhancement of Nile blue derivative-induced photocytotoxicity by nigericin and low cytoplasmic pH.

The mechanism of photocytotoxicity mediated by a lysosomotropic photosensitizer, Nile blue derivative (NBS-61), in relation to lysosome destruction was examined by lowering the intracellular pH with low extracellular pH and an ionophore, nigericin. The treatment performed after photoirradiation had minimal effect on the cytotoxicity. However, when the treatment was initiated before photoirradiation, it caused a three orders of magnitude enhancement on cytotoxicity with a two orders of magnitude enhancement by nigericin alone. This effect on cytotoxicity resembles closely that observed on photosensitization mediated by chloroaluminum phthalocyanine. The enhancement in this case has been attributed to the synergistic interaction between photodamage and perturbation of ion transports across mitochondrial or plasma membranes by nigericin. Because these are not the main sites of localization for Nile blue photosensitizers nor their initial targets of photocytotoxic action, data from the present study suggest the possibility of an intracellular dye translocation induced by nigericin, which redistributes the Nile blue photosensitizer from lysosomes to other sites, as a possible cause of the enhancement of cytotoxicity.

Cell Survival↗
PubMed1994

Novel CCK analogues and bombesin: a detailed analysis between phosphoinositide breakdown and high-dose inhibition of pancreatic enzyme secretion in three rodent species.

Cholecystokinin octapeptide (26-33) (CCK-8) stimulates pancreatic amylase secretion in a biphasic manner. Amylase secretion is stimulated in a dose-dependent manner up to a maximal level, but reduced secretion is observed at supramaximal concentrations. The downward portion of the dose-response curve has been referred to as "high-dose" inhibition. Recent studies with CCK-8 and Boc-Tyr(SO3H)-Nle-Gyl-Trp-Nle-Asp-2-phenylethylester (JMV-180) using rat acini have suggested that activation of the low-affinity CCK receptor leads to enhanced phosphoinositide (PI) breakdown, which in turn is responsible for high-dose inhibition of enzyme release. However, the secretory effect of JMV-180 varied considerably between rat and mouse. To explore further the relationship between PI breakdown and high-dose inhibition, we compared the effects of JMV-180 as well as the novel cholecystokinin tetrapeptide (30-33) Trp-Met-Asp-Phe-NH2 analogs A-70874 and A-57282, using rat, mouse and guinea pig pancreatic acini. The maximal secretory activity of CCK-8 was lowest (approximately 10% of total cellular amylase) in mouse, as compared with guinea pig and rat (approximately 15-20% of total amylase). The efficacies of A-70874, A-57282 and JMV-180 for stimulation of PI breakdown, relative to CCK-8, were 100, 100 and 45%, respectively, in mouse; 95, 70 and 20%, respectively, in rat and 75, 40 and 0%, respectively, in guinea pig.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗
PubMed1993

Regulation of manganese superoxide dismutase and other antioxidant genes in normal and leukemic hematopoietic cells and their relationship to cytotoxicity by tumor necrosis factor.

Myeloid cells are a major source of superoxide and other oxygen metabolites. As a protective mechanism, cells express antioxidant enzymes including manganese superoxide dismutase (Mn-SOD), copper-zinc SOD (Cu/Zn-SOD), and glutathione peroxidase (GSX-PX). Even though hematopoietic cells are a major source of oxidants, little is known of their expression of antioxidants. We found that seven myeloid leukemic cell lines blocked at different stages of differentiation constitutively expressed Mn-SOD, Cu/Zn-SOD, and GSX-PX RNAs. Level of Mn-SOD activities paralleled levels of Mn-SOD RNA. Terminal differentiation of native HL-60 cells to either granulocytes or macrophages did not alter levels of Mn-SOD RNA but markedly decreased cell division. Myeloid leukemic lines sensitive to cytotoxic effects of tumor necrosis factor (TNF) as well as normal peripheral blood lymphocytes and monocytes, dramatically increased their levels of Mn-SOD RNA in the presence of TNF. In contrast, Cu/Zn-SOD and GSX-PX RNA levels did not increase in these same cells. TNF-resistant leukemic lines had higher constitutive levels of Mn-SOD RNA and activity; and these levels did not change in the presence of TNF. Antisense but not random oligonucleotides to Mn-SOD markedly increased the sensitivity to the inhibitory effects of TNF for both the native HL-60 (TNF-sensitive) and K562 (TNF-resistant) cell lines. Further studies showed that the antisense oligonucleotides entered the cells and resulted in decreased levels of Mn-SOD RNA. The data suggest that Mn-SOD may provide protection against cytotoxicity of TNF in hematopoietic cells.

Base Sequence↗
PubMed1993

Peripheral cholecystokinin type A receptors mediate oxytocin secretion in vivo.

Cholecystokinin-octapeptide (CCK8) administered intraperitoneally (i.p.) in rats induces a rapid elevation in serum oxytocin (OT). The receptor subtype mediating this action of CCK was investigated with selective CCK-A and CCK-B receptor agonists and antagonists. CCK8 and A-71623, a potent CCK-A selective agonist, were similar in efficacy and potency for stimulating OT secretion. Both compounds at 10 nmol/kg elicited approximately one-half the response of 100 nmol/kg, which elevated serum OT to approx. 20 to 30-fold above basal level. The potent CCK-B selective agonist, A-63387, at doses up to 100 nmol/kg did not increase serum OT. MK-329, a CCK-A receptor selective antagonist, at a dose of 20 nmol/kg fully inhibited the action of 20 nmol/kg CCK8, while 100 nmol/kg of (R)L-365,260, a CCK-B selective antagonist, had no effect on the CCK8 response. These results, together with previous lesion studies, suggest that vagal CCK-A receptors in the periphery mediate the activation of the oxytocinergic pathway in vivo.

Animals↗
PubMed1993

The role of AP-1 in matrix metalloproteinase gene expression.

In an effort to understand the mechanism of matrix metalloproteinase (MMP) induction, lapine synoviocytes were isolated and incubated with phorbol myristate acetate (PMA) and autocrine "cell-activating factors" (CAF), agents which significantly increase MMP mRNA abundance. AP-1 complexes, formed by c-fos and c-jun which bind to 5' residues of the MMP genes, seem causally related to MMP gene expression in response to PMA. However, although AP-1 DNA binding activity is strongly induced following exposure of synoviocytes to CAF, MMP gene expression in response to CAF does not correlate well with AP-1 activity and is not inhibited by antisense DNA to fos and jun. We hypothesize that there is a CAF-response factor involved in MMP gene expression and that this factor competes with the binding of the AP-1 complex to its target response element.

Animals↗
PubMed1993

Double bond isosteres of the peptide bond: synthesis and biological activity of cholecystokinin (CCK) C-terminal hexapeptide analogs.

New and existing methodologies were used to prepare a series of modified CCK analogs in which each amide bond was replaced by a trans-alkene unit. The data indicate that every amide linkage at C-terminal tetrapeptide (CCK-4) region is crucial for biological activity. While the amide bond beyond the Trp residue in the N-terminal direction can be replaced by a trans-alkene and still retain most of the binding potency and functional activity.

Amino Acid Sequence↗
PubMed1993

Photodynamic destruction of lysosomes mediated by Nile blue photosensitizers.

Previous studies have established that a number of Nile blue derivatives are potent photosensitizers and that they are localized primarily in the lysosomes. The present study examines whether the lysosome is a main target of the photocytotoxic action mediated by these sensitizers. Chosen for this study were NBS-6I and sat-NBS, which represented, respectively, derivatives with high and moderate degrees of lysosomal. This is indicated by the light-and drug-dose-dependent losses of acid phosphatase staining particles, reduction of hexosaminidase in the lysosome-containing subcellular fraction, and impairment of the lysosomes to take up and sequester acridine orange. Ultrastructurally, swollen and ruptured lysosomes were seen as one of the first evidences of cell damage mediated by these photosensitizers. However, the study also showed that sat-NBS, which is less lysosomal selective, was less effective in mediating lysosomal destruction. Also, the degree of lysosomal destruction mediated by sat-NBS did not parallel the degree of cytotoxicity generated. This implies that for derivatives that are not exclusively localized in the lysosome, other subcellular sites may also be damaged by the photodynamic action and may play a role in the photocytotoxic process.

Acid Phosphatase↗